This application will provide training for Dr. Steven Bark in the neurobiology and neurochemistry of peptide neurotransmission using analytical and chemical technologies. With Dr. Bark's extensive experience in analytical and protein chemistry, this K01 training will focus on the neuroscience of peptides for neurotransmission. This training will occur under the mentorship of Dr. Vivian Hook, a noted researcher in protease processing in neurological systems. Dr. Hook's laboratory is in the Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California at San Diego. This institution and nearby allied institutions provide extraordinary opportunities for seminars, courses and collaborative interactions in all areas of Neuroscience. This training in peptide mechanisms of neurotransmission will enhance Dr. Bark's capabilities to identify and answer important questions about regulation of the nervous system in health and human disease as an independent research scientist. Enkephalin and beta-endorphin are opioid neuropeptides that have been implicated in adverse neurological disease conditions for mental state, chronic pain, drug and alcohol abuse, behavior, anxiety, and depression. The biosynthesis of these opioid peptides requires proteolytic processing of precursor proteins. Secretory vesicle Cathepsin L has recently been discovered as a new and significant protease pathway for production of enkephalins and beta-endorphin in vivo. These new findings indicate a distinct cysteine protease pathway, in addition to the well known subtilisin-like Prohormone Convertases 1 and 2 for production of neuropeptides. It is now important to define the role of Cathepsin L in producing these opioid neuropeptides. Therefore, the objective of this proposal is to define the protease mechanisms mediated by Cathepsin L for producing enkephalin and beta-endorphin. In Specific Aim 1, we will establish the identification and levels of enkephalin, beta-endorphin and their processing intermediates by coexpression of Cathepsin L and proenkephalin or proopiomelanocortin in PC12 cells. We will also optimize current mass spectrometry (MS) approaches for qualitative and quantitative analysis of neuropeptides. In Specific Aim 2, we will extend these experiments to evaluate Cathepsin L knockout mice for altered proteolysis of neuropeptides and precursor-derived intermediates that lead to production of enkephalin and beta-endorphin in vivo. In Specific Aim 3, we will define the changes in these neurotransmitters and their processing intermediates after stimulated secretion or activation in PC12 and primary chromaffin cells in culture. These mass spectrometry experiments will provide (1) significant advances for definitive identification and quantitation of neuropeptides and (2) use this critical information for defining proteolytic mechanisms for the biosynthesis of active enkephalin and beta-endorphin in health and in neurological disease conditions.